Pseudomonas aeruginosaInternalization by Human Epithelial Respiratory Cells Depends on Cell Differentiation, Polarity, and Junctional Complex Integrity

Abstract

Internalization of Pseudomonas aeruginosa by epithelial respiratory cell lines has been suggested to be dependent on the cystic fibrosis transmembrane conductance regulator (CFTR) protein. Because we have observed intracellular (IC) P. aeruginosa only in cells that do not express apical CFTR, we addressed the question of whether bacterial internalization by epithelial cells depends on the degree of cell differentiation and polarity. Internalization of piliated P. aeruginosa PAO-1 and PAK by human epithelial respiratory cells in primary culture and by the 16 human bronchial epithelial 14o- cell line cultured either on thick collagen gels or on thin collagen films was evaluated by the gentamicin exclusion assay. Cells cultured on thick gels were differentiated, polarized, and tight. They exhibited CFTR at their apical membranes, expressed beta1 integrins at their basal membranes, excluded lanthanum nitrate, and uniformly expressed ZO-1 protein. In contrast, in cells cultured on thin films, CFTR was present mainly in the cytoplasm, whereas beta1 integrins were detected at apical membranes. Most cells cultured on thin films did not exclude lanthanum nitrate and rarely expressed ZO-1 protein. Cells grown on thick and thin collagen substrates differed markedly in bacterial internalization: no IC bacteria could be detected in cells cultured on gels, whereas high IC bacterial concentrations were isolated from cells cultured on thin films. Treatment of cells cultured on thin films with ethylenediaminetetraacetic acid, to disrupt intercellular junctions further, significantly enhanced P. aeruginosa internalization. Our results suggest that P. aeruginosa internalization by epithelial respiratory cells does not depend on CFTR protein expression at the epithelial cell surface but rather on cell polarity and junctional complex integrity.

Role of Cystic Fibrosis Transmembrane Conductance Regulator in Pulmonary Clearance of Pseudomonas aeruginosa In Vivo

“…The distinctive bacterial penetration into interstitial sites and localization at basal surfaces of the bronchiolar epithelia are consistent with previous in vitro studies with nonmucoid strains that implicated PA virulence with access and binding of PA to the basolateral epithelial membrane (36, 37, 39, 49 -51). Invasion of PA bacteria to the basolateral surface in vitro is thought to occur through weakened tight junctions during injury-induced epithelial repair or at free epithelial edges in tissue culture epithelia (37). In this in vivo study, we found patches of bronchiolar epithelial cell erosion that could provide entry sites for bacterial invasion into basolateral and interstitial compartments.…”

Pseudomonas aeruginosa Acquires Biofilm-Like Properties within Airway Epithelial Cells

“…The gentamicin survival assay was used to detect intracellular bacteria based on the inability of this agent to penetrate epithelial cells to achieve sufficient concentrations to kill intracellular bacteria (13,24,31). Following incubation with bacteria, MTEC on supported membranes were washed twice for 5 min with sterile PBS, incubated with 50 g/ml gentamicin for 30 min, and washed twice again.…”

“…In the lung, discovery of intraepithelial Haemophilus influenzae in biopsy samples of airway epithelial cells from individuals with chronic obstructive lung disease harboring persistent antibiotic-resistant organisms implicates intracellular bacteria as an additional reservoir for infection (3,26). Experimental studies using cell lines and animal models of acute lung infection showed that P. aeruginosa can move intracellularly soon after infection (16,24,31,38,47). Most recently, in vitro studies using airway epithelial cell lines demonstrated that these bacteria are capable of intraepithelial survival for up to 24 h without cytotoxicity (10).…”

Cathelicidin LL-37 Increases Lung Epithelial Cell Stiffness, Decreases Transepithelial Permeability, and Prevents Epithelial Invasion by Pseudomonas aeruginosa

“…It is generally assumed that P. aeruginosa-induced lung inflammation involves binding to airway cells and subsequent direct and indirect cytotoxic effects (14,15). One possible result of bacteria-lung epithelial cell interaction is bacterial internalization (16), although the mechanism of entry and the role of intracellular organisms in the epithelial life cycle remain unclear (17)(18)(19)(20)(21). It was proposed that bacterial uptake might result in desquamation of infected cells and bacteria clearance (14), intracellular survival of bacteria without killing the host cells, and host cell death through bacteriamediated cytotoxicity (18).…”